PROJECT SUMMARY/ABSTRACT A large accumulation of evidence has implicated estrogen in breast cancer development. One way in which estrogen impacts breast cancer is via the catechol estrogen (CE) metabolism pathway. Particular CE metabolites, namely hydroxy estrogen quinones, are capable of forming DNA adducts which may result in DNA mutations. Recently, Cavalieri and colleagues developed an assay to identify estrogen DNA adducts (EDA) in urine samples. These researchers observed that a ratio of EDA to their unbound counterparts (estrogen metabolites and conjugates [EMC]) was higher in breast cancer patients and those women at high risk of breast cancer compared to low-risk, healthy controls. Additional studies demonstrated that the EDA:EMC ratio was reduced in response to antioxidants. This biomarker represents a potential marker for use in clinical interventions, as well as in prospective, epidemiologic studies aiming to replicate the early findings on the EDA:EMC ratio and breast cancer. However, more research is needed on the EDA:EMC ratio before we move to investigate the EDA:EMC ratio in large-scale studies. First, a reliability study of the EDA:EMC ratio is needed in order to indicate the level of variabiity in the EDA:EMC ratio over time. We will conduct a reliability study to investigate the variability of the EDA:EMC ratio within and between individuals over time. Specifically, we propose to recruit 33 women (of which we anticipate 26 will complete the study) in whom we will collect 8 repeated urine samples in order to calculate the inter- and intra-person variability of the EDA:EMC ratio (Specific Aim 1). As part of the proposed study, we will also explore the influence of health and demographic factors on the EDA:EMC ratio (Secondary Aim 1), as well as to examine the impact of menstrual cycle phase on the EDA:EMC ratio (Secondary Aim 2). Urinary EDA and EMC will be measured by ultraperformance liquid chromatography/tandem mass spectrometry and metabolite concentrations will be used to calculate ratios of EDA:EMC. The proposed study would be the first reliability study of the EDA:EMC ratio. The results of this investigation will provide important information on the variability of the EDA:EMC ratio and its potential use as a biomarker in breast cancer research. Data emanating from this proposal will be particularly useful in the development of large, prospective epidemiologic studies of breast cancer and intervention trials aimed at breast cancer prevention.